Most prion strains infect a relatively small number of animal species, but the strain responsible for bovine spongiform encephalopathy (BSE) and variant Creutzfeldt Jakob disease (vCJD) is remarkably promiscuous. The BSE/vCJD strain has caused epidemics in a wide variety of animal species, including humans, but it is unclear how this strain originated and why it is particularly adept at crossing species barriers. Here, we propose to study how promiscuous prion strains originate and identify the molecular mechanism responsible for their ability to cross species barriers easily. We recently reported that cofactor molecules regulate the major strain properties of mammalian prions, including neurotropism and PrPSc conformation. Building upon this critical result, we will now study whether cofactor molecules also play a role in cross- species transmission of prions. Our hypothesis is that prions originally formed with a particular cofactor will be more likely to cross a species barrier if that cofactor is available by modulating the potential folding pathways for PrP molecules. We will also characterize and isolate the cofactor molecule(s) responsible for maintaining the strain properties of the BSE/vCJD strain, including its ability to infect multiple animal species. To do this, we will employ a simple reconstitution assay in which maintenance of the BSE/vCJD strain PrPSc conformation can be monitored rapidly. Finally, we will test the hypothesis that promiscuous prion strains are created from non- promiscuous strains as a result conformational selection during the commercial heat rendering process used to recycle animal carcasses. To test this hypothesis, we will simulate the rendering process in the laboratory and study whether sheep scrapie and deer CWD can be converted into promiscuous strains capable of infecting humans or cows. These studies will have an immediate impact on public health.